Numerous recombinantly produced proteins have received regulatory approval for human therapeutic use. A growing number of these therapeutic proteins are produced in microbial expression systems. Indeed, according to a recent review, microbial expression systems are used for production of nearly half of the 151 protein-based recombinant pharmaceuticals licensed up to January 2009 by the U.S. Food and Drug Administration or European Medicines Agency (Ferrer-Miralles et al., Microbial Cell Factories 2009, 8:17).
Among these recombinantly produced proteins are antibodies, which conventionally are tetrameric proteins composed of two identical light chains and two identical heavy chains. Hundreds of therapeutic monoclonal antibodies (mAbs) are currently either on the market or under development. The production of functional antibodies generally involves the synthesis of the two polypeptide chains as well as a number of post-translational events, including proteolytic processing of the N-terminal secretion signal sequence; proper folding and assembly of the polypeptides into tetramers; formation of disulfide bonds; and typically includes a specific N-linked glycosylation.
Fungal hosts such as the methylotrophic yeast Pichia pastoris have distinct advantages for therapeutic protein expression, including that they do not secrete high amounts of endogenous proteins, have strong inducible promoters available for producing heterologous proteins, can be grown in defined chemical media and without the use of animal sera, and can produce high titers of recombinant proteins (Cregg et al., FEMS Microbiol. Rev. 24: 45-66 (2000)). Prior work, including work conducted by the present inventors, has helped established P. pastoris as a cost-effective platform for producing functional antibodies that are suitable for research, diagnostic, and therapeutic use. See co-owned U.S. Pat. Nos. 7,927,863 and 7,935,340, each of which is incorporated by reference herein in its entirety. Methods are also known in the literature for design of P. pastoris fermentations for expression of recombinant proteins, with optimization having been described with respect to parameters including cell density, broth volume, pH, substrate feed rate, and the length of each phase of the reaction. See Zhang et al., “Rational Design and Optimization of Fed-Batch and Continuous Fermentations” in Cregg, J. M., Ed., 2007, Pichia Protocols (2nd edition), Methods in Molecular Biology, vol. 389, Humana Press, Totowa, N.J., pgs. 43-63, which is hereby incorporated by reference in its entirety.
Additionally, prior work by the present applicants and others has described increasing production of proteins in yeast through methods including addition of a bolus of ethanol to the culture at or near the beginning of the production phase, and with respect to multi-subunit proteins such as antibodies, varying the number of gene copies and the copy number ratio between subunit genes. See US20130045888, entitled, Multi-Copy Strategy For High-Titer And High-Purity Production Of Multi-Subunit Proteins Such As Antibodies In Transformed Microbes Such As Pichia Pastoris; and US20120277408, entitled, High-Purity Production Of Multi-Subunit Proteins Such As Antibodies In Transformed Microbes Such As Pichia Pastoris. 
Though the aforementioned Zhang et al. article makes some effort to describe a systematic approach to optimizing the aforementioned parameters and give a theoretical approach to understanding the interplay between some of these parameters, expression optimization remains a largely empirical process. Because of this interplay, it is generally insufficient to optimize each individual parameter while keeping all others constant. For example, optimal media composition may vary with culture density, strain background, feed rate, agitation, oxygenation, etc. Because of this complex interplay, the number of combinations of parameters that can be tested is potentially infinite, and even if an expression system has been extensively optimized there always remain a large number of untested conditions which could benefit yield and/or purity.